Therapeutic monoclonal antibodies (mAbs) have met some successes in the clinic over the last years, particularly in oncology. More than twenty five mAbs are on the market.
Many technical efforts have been made to generate second generation mAbs with decreased immunogenicity and with optimized effector functions. Since the majority of therapeutic antibodies are IgG1, at least part of the observed in vivo effects of mAbs might be induced following interactions between their Fc region and FcγR. Notably, the ability of mAbs to kill tumor cells has been related to their capacity to recruit and activate effector cells such as NK cells and macrophages through receptors for the Fc portion of IgG (FcγR).
However, recent reports have shown that the efficiency of IgG1 human therapeutic mAbs is strongly affected by various parameters: changes in Fc glycosylation, FcγRIIIA polymorphism, interaction with inhibitory FcγRIIB, and competition with endogenous IgG for FcγRI and FcγRIII binding. For instance, studies with FcγR−/− mice have revealed the implication of different FcγR in some in vivo mechanisms of action of two widely used therapeutic mAbs, trastuzumab and rituximab. These cytotoxic mAbs directed against tumors engage both activating (FcγRIIIA) and inhibitory (FcγRIIB) receptors. In these studies, a more pronounced tumor regression was observed in FcγRIIB-deficient mice than in wild-type mice, whereas FcγRIIIA-deficient mice were unable to stop tumor growth in the presence of therapeutic mAbs. In humans, a recent study has shown that the therapeutic efficiency of rituximab (a chimeric human IgG1) in patients with non-Hodgkin lymphoma is partly correlated with FcγRIIIA polymorphism. Thus, patients homozygous for the Val158 allele (IgG1 high binder) exhibited a higher response to the treatment than the patients homozygous for the Phe158 allele (IgG1 low binder). Similarly, engineered IgG glycoforms have been shown to trigger optimized ADCC through the recruitment of FcγRIIIA. A first study showed that an IgG1 antibody engineered to contain increasing amounts of bisected complex oligosaccharides (bisecting N-acetylglucosamine, GlcNAC) allows the triggering of a strong ADCC as compared to its parental counterpart. Second, a lack of fucose or low fucose content on human IgG1 N-linked oligosaccharides has been shown to improve FcγRIIIA binding and ADCC as well as to increase the clearance rate of Rhesus D+ red blood cells in human volunteers. Moreover, it has been recently shown that the antigenic density required to induce an efficient ADCC is lower when the IgG has a low content in fucose as compared to a highly fucosylated IgG.
The idea that a better control of Fc/FcγR interactions was needed when using therapeutic mAbs has been clearly argued in the early 80's and led to the concept and the generation of bispecific antibodies (bsAbs), using biochemical approaches and then molecular engineering in the early 90's. Bispecific antibodies, able to bring together target cells and activated effector cells have important potential advantages over whole naked mAbs. Notably, with regard to NK cells recruitment and activation, bsAbs make it possible to overcome most of the problems encountered with therapeutic mAbs. First, it is far easier to use an antibody arm binding to FcγRIIIA than to engineer and fine-tune the interaction between the antibody Fc region and FcγRIIIA. It is indeed possible to select a FcγRIIIA binder devoid of cross reaction for inhibitory FcγRIIB and targeting an epitope not involved in the Fc binding to avoid the high/low binder FcγRIIIA polymorphism issue, as well as endogenous IgG competition. Moreover, antibody fragments are not concerned by glycosylation issues, and it is possible to fine-tune the affinity of the antibody from a μM to a nM range, i.e., an affinity up to 1,000 fold higher than that involved in Fc/FcγRIIIA interaction. Thus, a number of attempts have been made to create anti-FcγRIIIA×anti-target bsAbs.
However, for years, these attempts were hindered by the impossibility to efficiently produce such molecules, the most efficient techniques requiring grams of mAbs to produce milligrams of heterogeneous preparations of bsAbs. Therefore, the first generation of bsAbs never reached the market, mostly due to the cost of getting molecules with bi-functional properties in large amounts for a therapeutic use.
The inventors have recently developed a new generation of bispecific antibodies, based on llama VHH (sdAb for single domain antibody or also Nb for nanobody), that can be easily produced in E. coli and that overcome the limitations listed above, while being able to exert a strong tumor lysis at extremely low concentrations. These bispecific antibodies are described in the International Patent Application no WO/2006/064136. This generation of therapeutic antibodies has the potential to rapidly translate into efficient therapeutics. Although these bsAbs accumulate within the tumor, they suffer from a rapid elimination due to their relatively small size, below the renal threshold (around 60 kDa), and to the absence of Fc region involved in the interaction with the FcRn receptor, responsible for the long serum half-life of full length IgG. Thus, there is a need to improve these bsAbs in terms of efficiency, serum half-life and biodistribution.